Subcutaneous administration of casein attenuates atherosclerotic progression in male apoE‐/‐ mice fed with high‐fat diet

Abstract The impact of casein on atherosclerotic lesion progression remains controversial. In this study, we tested the effect of casein on atherosclerotic development and its potential mechanisms in male apolipoprotein E knockout (apoE‐/‐) mice fed with high‐fat diet (HFD). Male apoE‐/‐ mice fed with HFD were randomized into HFD group (subcutaneous injection with 0.5 ml of 0.9% sodium chloride daily, n = 6) and HFD + Casein group (subcutaneous injection with 0.5 ml of 10% casein daily, n = 6). Body weight was recorded at baseline and once a week thereafter. After 12 weeks of treatment, plasma lipid and inflammatory markers, and histological characterization of atherosclerotic plaques in the aortic arch and aortic sinus were analyzed. There was no significant difference in weight gain between the two groups after 12 weeks of treatment. Plasma levels of total cholesterol (TC), triglyceride (TG), and low‐density lipoprotein cholesterol (LDL‐C) were significantly lower, while high‐density lipoprotein cholesterol (HDL‐C) level tended to be higher in the HFD + Casein group compared with the HFD group. The positive percentages of atherosclerotic lesions in aortic arch and aortic sinus as well as collagen deposition in aortic sinus plaques were significantly lower in the HFD + Casein group compared with the HFD group. Plasma levels of interleukin (IL)‐1β and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were also significantly lower in the HFD + Casein group compared with the HFD group. In conclusion, subcutaneous administration of casein attenuates atherosclerotic lesion progression, possibly through decreasing fibrosis and inflammatory responses in male apoE‐/‐ mice fed with HFD.

Casein comprises approximately 80% of the total milk protein (Dalziel, Young, McKenzie, Haggarty, & Roy, 2017), which is not only naturally present in dairy, but also popularly utilized as a protein supplement in its purified form of various nutrition products. Casein is often applied as an animal source of protein in experimental research as well. Till now, the role of casein in human health, especially on the lipid metabolism remains an issue of debate. Huff, Roberts, and Carroll (1982) found that casein enhanced triglyceride (TG) levels in plasma and atherosclerotic lesions development in the aortic arch region in male New Zealand white rabbits. Similarly, Damasceno et al. (2001) reported that casein feeding contributed to increasing plasma cholesterol and TG concentrations and the area of aorta atherosclerotic lesions in adult male New Zealand rabbits, while Ijaz et al. (2018) showed that casein diet for 12 weeks did not affect the lipid profile in male C57BL/6J mice when consumed with either low-fat diet (LFD) or high-fat diet (HFD). Another study conducted by Meinertz, Nilausen, and Faergeman (1990) suggested that the low-cholesterol or cholesterol-enriched casein diets could improve plasma lipid concentrations in healthy participants.
It is well known that inflammation is a key factor in the progression of atherosclerosis (Hedin & Matic, 2018). It was demonstrated that chronic systemic inflammation, characterized by the increase of serum levels of C-reactive protein (CRP) and proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin (IL)-6, was closely related to pathogenesis of multiple metabolic disorders including insulin resistance, T2DM, obesity, and dyslipidemia in both humans and animal models (Burhans, Hagman, Kuzma, Schmidt, & Kratz, 2018;Cox, West, & Cripps, 2015;Lira, Rosa Neto, Antunes, & Fernandes, 2014;Lontchi-Yimagou, Sobngwi, Matsha, & Kengne, 2013). Studies over the last several decades suggested that milk proteins had immunomodulatory effects but the results were mixed. It was reported that κ-casein suppressed lymphocyte proliferation induced by T-and B-cell mitogens (Cross & Gill, 2000). Panagiotakos, Pitsavos, Zampelas, Chrysohoou, and Stefanadis (2010) found a decreased level of inflammatory markers associated with the consumption of dairy products among healthy adults. However, Pal and Ellis (2010) demonstrated that casein supplementation had no significant influence on plasma inflammatory markers in overweight and obese individuals. This finding is in accordance with the study conducted by Bohl, Bjornshave, Gregersen, and Hermansen (2016) which showed that casein protein did not alter circulating inflammatory markers in abdominally obese adults. Till now, the effect of casein on atherosclerotic progression and its potential mechanism are not fully understood. In this study, we tested the impact of casein on the atherosclerotic development in apolipoprotein E knockout (apoE -/-) mice fed with HFD (1.25% cholesterol).

| Animals and study groups
Six-week-old male apoE -/mice on C57BL/6 background were purchased from The Animal Center of Beijing University and kept under standard laboratory conditions (12 hr light cycle, temperature 25°C), with free access to standard chow and drinking water. After 2 weeks of acclimatization, the mice were weighed and randomly divided into HFD group (n = 6, subcutaneous injection with 0.5 ml of 0.9% sodium chloride daily) and HFD + Casein group (n = 6, subcutaneous injection with 0.5 ml of 10% casein daily) and fed with HFD for 12 weeks. Male mice were chosen in this study to avoid the potential impact of periodic sex hormone fluctuation on atherosclerosis. Subcutaneous injection was used to evaluate the effect of casein in a fixed dose range.
This study was approved by the Animal Care Committees of Wuhan Fourth Hospital. Experimental protocol was based on the ARRIVE guidelines and guidelines from the National Institutes of Health for animal care and use (NIH Publications No. 85-23, revised 1996).

| Harvesting of tissue
Body weight was recorded at baseline and once a week thereafter for 12 weeks. After 12 weeks of treatment, the mice were anesthetized with pentobarbital sodium (40 mg/kg) after 6 hr of fasting.
Blood samples were collected from retro-orbital plexus into heparin-coated tubes and then centrifuged to separate plasma. Both the heart and whole aorta were rapidly removed. All samples and tissues were snap-frozen and stored at −80°C until analysis.
Plasma levels of IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed using the ELISA kits (Neobioscience, EMC111, Shenzhen, China) according to the manufacturer's protocols. Plasma CRP level was measured with the ELISA kit (Elabscience, Wuhan, China) following the manufacturer's instructions. The absorbance was recorded at 450 nm.

| Histological characterization of aortic arch and aortic sinus
Method used to evaluate the atherosclerosis in this model was identical as the method we used previously (Gao et al., 2015). Masson's Trichrome staining, respectively. Images were captured by microscope (Leica, DMi8, Germany) with 40 magnifications, and the stained area was measured using IPP software.

| Statistical analysis
Data were expressed as mean ± standard deviation (SD). Normal distribution was assessed by the Kolmogorov-Smirnov test. Unpaired Student's t test was used to evaluate statistical differences between groups. A value of p < .05 was considered statistically significant. All analyses were performed using SPSS 21.0 software (SPSS Inc., Illinois, USA).

| Body weight and plasma lipid parameters of HFD group and HFD + Casein group
Baseline and final body weight as well as body weight gain were similar between the HFD group and HFD + Casein group (Table 1, Figure 1). Plasma levels of TC, TG, and LDL-C were significantly lower, while HDL-C level tended to be higher in the HFD + Casein group compared with the HFD group (Table 1).

| Histological characterization of aortic arch and aortic sinus of HFD group and HFD + Casein group
Histological characterization of atherosclerotic plaques in the aor-

| Plasma levels of inflammatory markers of the HFD group and HFD + casein group
As shown in Figure 3, plasma levels of IL-1β and GM-CSF were significantly lower, and IL-6 and CRP tended to be lower in the HFD + Casein group compared with the HFD group. F I G U R E 1 Body weight at each week for 12 weeks. ApoE -/mice were fed with high-fat diet for 12 weeks without (HFD group) or with 10% casein injections (HFD + Casein group). The body weight was measured once a week for 12 weeks

| D ISCUSS I ON
The major findings of this study are as follows: (1)

| The influence of casein on blood lipid level in male apoE -/mice fed with HFD
Atherosclerosis remains the major cause of ischemic vascular diseases. Since the underlying pathological process of ischemic  (Brown & Goldstein, 1983).
Second, gender appeared to play a role on blood lipid levels and the development of atherosclerosis. Terpstra, Van Tintelen, and West (1982)  reported to be larger and more advanced in young female than male apoE -/mice because of sex differences in immune mechanisms with activated T cells responding to oxidized LDL in female mice even in the absence of exogenous estrogens, while no such effect was seen in male mice (Caligiuri, Nicoletti, Zhou, Tornberg, & Hansson, 1999).
These studies indicated that female animals were more predisposed to developing hypercholesterolemia and atherosclerosis in response to casein diet than their male counterparts.
Third, serum lipid level is quite sensitive to the protein level in the diet. A study in female lean Zucker strain rats showed that the hypercholesterolemic effect of dietary casein could be enhanced by increasing the proportion of this protein in the diet from 20% to 50%. (Terpstra et al., 1982) Furthermore, Huffman and Jones (1956) observed that the level of serum cholesterol in male weanling rats of the Sprague-Dawley stock fed 12.5% casein for 25 weeks was significantly lower than in those fed 25%, 9%, or 7.5% and tended to be lower than in those fed 18.75%, which revealed that reduction or elevation of dietary casein beyond a modest concentration (12%-18%) might lead to ultimate elevation of serum cholesterol. This observation was supported by Fillios et al. (1958) (Kersten, 2014). A study in male Wistar rats confirmed that tissue LPL activities were decreased with the consumption of a low-protein diet containing 50 g/kg protein compared with a balanced diet containing 200 g/kg protein (Boualga, Bouchenak, & Belleville, 2000).As a consequence, inadequate levels of dietary protein followed by decreased LPL activity could increase the levels of TG in plasma. These studies explained that too high or too low proportion of casein in dietary could increase the levels of serum lipids.
Taken together, the results from above studies might partially explain inconsistent effects of casein on serum cholesterol levels.
Therefore, it was reasonable for 10% casein to reduce blood lipid levels in male apoE -/mice fed with HFD in this study. Our study provides a potential way to alleviate hyperlipidemia induced by HFD in some individuals through administration of appropriate casein proportion.

| Potential mechanisms of casein affecting atherosclerotic progression
Vascular fibrosis, involving accumulation of extracellular matrix proteins particularly collagen, is associated with atherosclerosis. Previous study showed that vascular fibrosis contributed to vascular remodeling and scar formation (Touyz, 2005). Arterial stiffening or remodeling was recognized as an important consequence of vascular fibrosis to promote the development of atherosclerosis (Shirwany & Zou, 2010

| Study limitations
The influences of casein on atherosclerotic development are varied in different gender and doses. This study only demonstrated that casein (subcutaneous injection with 0.5 ml of 10% casein daily for 12 weeks) attenuated atherosclerotic development in male apoE -/mice fed with HFD (1.25% cholesterol). Future studies are warranted to explore dose dependent effect of casein on atherosclerotic progression and its potential mechanism in both male and female apoE -/mice. It is to note that present study gave casein to mouse by subcutaneous injection, which is different from previous studies and is also different from casein's daily oral consumption habits; therefore, the results of subcutaneous injection cannot be compared with the results of previous oral consumption.
In conclusion, our study finds that 10% casein treatment decreases lipid concentrations in male apoE -/mice fed with HFD.
Casein administration is associated with attenuated atherosclerotic lesion progression, possibly by decreasing fibrosis and inflammatory responses in male apoE -/mice fed with HFD.

| E THI C AL RE VIE W
This study was approved by the Animal Care Committees of Wuhan Fourth Hospital.

ACK N OWLED G M ENT
This work was supported by the Nature Science Foundation grant of Hubei 2018CFB61 (Hubei Province, China).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.